PROJECT SUMMARY Clostridium difficile is a major public health threat, causing disease ranging from mild diarrhea to pseudomembranous colitis. C. difficile disease symptoms are largely mediated by the secreted cytotoxins, TcdA and TcdB. Many aspects of the pathogenicity of this bacterium remains poorly understood, including how C. difficile regulates the production of the toxins and other virulence factors. Toxin gene expression is linked to expression of flagellar genes via the flagellar sigma factor sigma-D, which is encoded in the flgB operon. Thus, factors that regulate the expression of the flgB operon will impact toxin production in addition to flagellar motility. Our long-term goal is to determine how C. difficile coordinately regulates flagellum and toxin gene expression, as these mechanisms are likely fundamental to pathogenesis. We recently showed that flagellar gene expression is subject to phase variation through site-specific DNA recombination of an invertible sequence upstream of the flgB operon. Notably, inversion of the DNA sequence, which we term the ?flagellar switch?, also impacts the production of toxins essential for virulence. We identified RecV as the DNA recombinase that mediates inversion of the flagellar switch, and we have begun to define the mechanism by which the orientation of the flagellar switch sequence controls gene expression. Our central hypothesis is that phase variation of flagellum and toxin production is critical to C. difficile pathogenesis, and consequently interfering with the ability of C. difficile to phase vary will attenuate one or more aspects of virulence. While flagella and/or motility may be important for some aspects of infection, downregulation of flagella is likely vital for C. difficile to evade host immune recognition. Toxin production may similarly be beneficial or detrimental to C. difficile during different stages of infection. The objective of this proposal is to define the molecular mechanisms underlying phase variation and to evaluate the impact of phase variation on host colonization and virulence. This proposal is innovative because it represents an entirely new area of investigation toward understanding C. difficile disease: phase variable expression of flagella and toxins. Completion of the proposed studies will provide much needed mechanistic information on how C. difficile controls virulence factor production during infection, and may expose potential targets for inhibition of intestinal colonization and toxin production to facilitate efforts to combat this increasingly problematic pathogen.